Identification of Cdna Clones Encoding a Precursor of Rat Liver
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 2320-2324, April 1985 Biochemistry Identification of cDNA clones encoding a precursor of rat liver cathepsin B (proteolytic processing/oligonucleotide probes/thiol proteases/lysosomes) BLANCA SAN SEGUNDO, SHU JIN CHAN, AND DONALD F. STEINER Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637 Contributed by Donald F. Steiner, December 17, 1984 ABSTRACT Recentstudies have suggested that many Iy- be involved in the processing of proinsulin to insulin (4, 5), sosomal enzymes, including cathepsin B (EC 34.22.1), may be while the mature forms of the enzyme may play a role in synthesized as larger precursors and proteolytically processed secretory granule turnover (3). to their mature forms.,To determine the structure of the pri- To more fully define the nature of the initial precursor of mary translation product of cathepsin B, we have screened a cathbpsin B, to investigate the processing events involved in phage.cDNA library fordclQnes encoding rat.liVer cathepsin B. generating intermediate and mature forms of the enzyme, We synthesized two extended DNA oligoniucleotides to use as and to gain more insight into those structural features of the hybridization probes: a 50-mner corresponding.to the coding precursor that might be related to the dual targeting of ca- segment for residues 215-231 of mature cathepsin B and a 54- thepsin B during its biosynthesis, we have cloned the ratliv- mer corresponding to residues 117-134. After screening er enzyme. Here we provide a report of the strategy that 600,000 plaques, five clones were obtained that hybridized to prdved successful and preliminary results on the character- the 32P-labeled 50-mer; of these, two (KrCB3 and XrCB5) also ization of two cloned cDNA fragments that encode the ma- reacted with the 54-mer.
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